Investigating the effect of Fusobacterium nucleatum on the aggressive behavior of cancer-associated fibroblasts in colorectal cancer

Fusobacterium nucleatum, (F. nucleatum) as a known factor in inducing oncogenic, invasive, and inflammatory responses, can lead to an increase in the incidence and progression of colorectal cancer (CRC). Cancer-associated fibroblasts (CAF) are also one of the key components of the tumor microenvironment (TME), which lead to resistance to treatment, metastasis, and disease recurrence with their markers, secretions, and functions. This study aimed to investigate the effect of F. nucleatum on the invasive phenotype and function of fibroblast cells isolated from normal and cancerous colorectal tissue. F. nucleatum bacteria were isolated from deep periodontal pockets and confirmed by various tests. CAF cells from tumor tissue and normal fibroblasts (NF) from a distance of 10 cm of tumor tissue were isolated from 5 patients by the explant method and were exposed to secretions and ghosts of F. nucleatum. The expression level of two markers, fibroblast activation protein (FAP), and α-smooth muscle actin (α-SMA), and the amount of production of two cytokines TGF-β and IL-6 from fibroblast cells were measured by flow cytometry and ELISA test, respectively before and after exposure to different bacterial components. The expression of the FAP marker was significantly higher in CAF cells compared to NF cells (P < 0.05). Also, the expression of IL-6 in CAF cells was higher than that of NF cells. In investigating the effect of bacterial components on the function of fibroblastic cells, after comparing the amount of IL-6 produced between the normal tissue of each patient and his tumoral tissue under 4 treated conditions, it was found that the amount of IL-6 production from the CAF cells of patients in the control group, treated with heat-killed ghosts and treated with paraformaldehyde-fixed ghosts had a significant increase compared to NF cells (P < 0.05). Due to the significant increase in FAP marker expression in fibroblast cells of tumor tissue compared to normal tissue, it seems that FAP can be used as a very good therapeutic marker, especially in patients with high levels of CAF cells. Various components of F. nucleatum could affect fibroblast cells differentially and at least part of the effect of this bacterium in the TME is mediated by CAF cells.


Introduction
Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer death in the world [1][2][3].Despite the existence of systemic treatments such as chemotherapy and radiotherapy, a large number of patients are facing disease progression due to resistance to treatment.Accordingly, the 5-year survival is less than 10% of the present study is a preliminary investigation regarding the effect of F. nucleatum on the function and phenotype of fibroblast cells isolated from the tumor environment and the adjacent normal tissue.

Bacterial isolation and identification
F. nucleatum was isolated from the periodontal tooth pockets in patients with chronic periodontal pockets who were referred to the Department of Periodontology, Faculty of Dentistry, Isfahan University of Medical Sciences between December 2020 and July 2021.Patients had taken antibiotics for at least one month before the sampling and had a pocket depth ≥ 5 mm.To remove normal oral flora, the surface of dental abscesses was wiped off with sterile gas and 70% ethyl alcohol.Then a paper point (size 40) was inserted deeply into the patient's periodontal pockets and held for 30 s.The samples were streaked on Columbia agar (CA) (CM0331, Oxoid, UK) supplemented with 5 µg/ml hemin, 10 µg/ ml vitamin K, 5% defibrinated sheep blood, 1 μg/ml norfloxacin (Sigma, USA), and 4 μg/ml vancomycin (Sigma, USA) and placed in an anaerobic jar containing type A GasPak (Anaerocult A, Merck KGaA, Germany) to create anaerobic conditions, and were transferred to the laboratory [43].
Cultures incubated at 37 ºC for 48 h and sub-cultured several times on CA supplemented with 5 µg/ml hemin, 10 µg/ ml vitamin K, and 5% defibrinated sheep blood, 1 μg/ml norfloxacin, and 4 μg/ml vancomycin under anaerobic conditions (N2:CO2:H2, 85∶5∶10).The growth on the plate was examined macroscopically and samples with colonies similar to F. nucleatum were gram-stained, and subsequently, biochemical tests were performed to confirm bacterial identity.In this regard, antibiotic sensitivity was carried out for colistin (10 µg/ml), penicillin (10 µg/ml), kanamycin (1000 µg/ ml), and vancomycin (5 µg/ml) using a CA plate without antibiotic, hemin, and vitamin K1.The plates were placed in an anaerobic jar at 37 ºC for 48 h and then checked visually for the presence or absence of the zone of inhibition.To perform the indole and lipase test, the isolates were cultured on Sulfide Indole Motility (SIM) (CM0435, Oxoid, UK) and Egg Yolk agar (EYA) (CM0131, Oxoid, UK) plate, respectively, and were incubated in an anaerobic jar at 37 ºC for 48 h.

Bacterial ghosts' and secretome preparation
For preparation of the soluble fraction from F. nucleatum, preserved bacteria were reconstituted in 100 ml BHI broth with 150 × 10 6 bacteria corresponding to 0.5 McFarland in anaerobic condition for 48 h.After centrifugation of the cultured medium, the supernatant was filtered through the 0.22 µm sterile filter and stored at −70.For the preparation of bacterial ghosts, the pellet was washed three times and dissolved in PBS and the concentration was determined at 600 nm.The prepared bacterial suspension was submitted to kill heat and Paraformaldehyde (PFA), respectively.For heat killing, 2 ml of bacterial suspension in PBS was subjected to a temperature of 100 °C for 10 min and stored at −70 °C.For PFA fixation, 2 ml of bacterial suspension in PBS was centrifuged and the bacterial pellet dissolved in 4 ml of 1% PFA.The solution was incubated for 30 min at room temperature in the dark.Then the solution was centrifuged, the ghost was washed twice, dissolved in 2 ml of PBS, and stored at −70.

Isolation of normal and cancer-associated fibroblasts (CAFs)
To isolate fibroblast cells, 20 patients with CRC who underwent surgery from September 2021 to April 2022 at the Alzahra Hospital (Isfahan, Islamic Republic of Iran), were included in the study.Patients, who were selected for sampling, had not undergone chemotherapy, radiotherapy, or immunotherapy before surgery and had no history of autoimmune diseases.Sampling was done in the operating room by a surgeon under the supervision of a pathologist to select CRC tissue and adjacent normal tissue at a distance of 10 cm from the tumor.Due to the limited life span of the isolated primary cells or contamination with bacteria or fungi, only samples from 5 patients [CAFs and normal fibroblasts (NF)] were obtained.Demographic characteristics of included subjects are summarized in (Table 1).
After transferring the tissues to the laboratory under sterile conditions, the samples were washed 3 times in Phosphatebuffered saline (PBS) containing 5% fetal bovine serum (FBS) and 3% penicillin/streptomycin antibiotics.Then, peripheral and necrotic tissues were removed, and the remaining tumor and normal tissues were fully cut up into 1 mm pieces and

Co-culture of fibroblast and F. nucleatum components
The co-culture experiments were carried out in conditions in which primary fibroblasts were adapted as described above.Briefly, fibroblast cells (passage 3) were seeded at 1 × 10 5 cells per well in 6-well plates containing 2 mL DMEM) without L-glutamine and cultured for 48 h in a humidified incubator at 37 °C and 5% of CO2.After that, cells were washed twice with PBS and new media containing the different components of the F. nucleatum, including secretome and ghost was added to infect the monolayer cells.There were 4 conditions for each extracted NF and CAF, including the cells without treatment as the control group, the cells treated with secretome in a 1:1 ratio in medium, the cells treated with the ghost of heat-killed bacteria, and the cells treated with the ghost of PFA-fixed bacteria.The F. nucleatum ghosts were added to the cells at a multiplicity of infection (MOI) of 100 and incubated for more than 48 h.

Flow cytometry analysis
After encountering CAF and NF cells with F. nucleatum fragments, the cells were harvested, re-suspended in PBS, and washed twice.To perform surface staining for FAP marker detection, cold PBS containing the mouse anti-human FAP antibody (1: 40, cat#FAB3715, R&D system, USA) was added.After 30 min of incubation at 4 ºC, the cells were washed with cold PBS.Then, cells were fixed with 4% formaldehyde for 15 min, permeabilized with cold 1 × Perm/wash (cat#421002, Biolegend, USA) for 30 min, and incubated with mouse anti-human α-SMA antibody (1: 20, R&D system, cat#ICI420P, USA) for 40 min in the dark.This was followed by centrifugation and cells were resuspended in PBS.All stained cells were analyzed by flow cytometry (BD Bioscience, USA).Analysis of the obtained results was performed using FlowJo v10.8 (FlowJo LLC, USA).

Enzyme-linked immunosorbent assay (ELISA)
After 48-h incubation of CAF and NF cells with F. nucleatum components in 6-well plates, fibroblast-conditioned media was collected and stored at −80 ºC for cytokine assay.Human IL-6 (Cat.430,504, Biolegend, USA) and human TGF-β (Karmania Pars Gene, Iran), were assessed using ELISA according to the manufacturer's protocol.Each sample was assayed in triplicate.Concentration was quantified by measuring the absorption at 450 nm with a microplate reader.

Quantification of F. nucleatum in colon tissue by real-time PCR
After surgical resection, the colorectal tumor and adjacent nonmalignant tissue samples were snap-frozen in liquid nitrogen and stored at −70 °C.Genomic DNA was extracted from the tissues using the phenol-chloroform method [44].
To quantify the amount of F. nucleatum DNA in each sample, real-time PCR was performed for the F. nucleatum nusG gene.The cycle threshold (Ct) values for F. nucleatum were normalized to the amount of human biopsy gDNA in each reaction by using the reference gene, prostaglandin transporter (PGT), too.

Statistical analysis
Statistical analyses were performed using GraphPad Prism software version 9.4.1.Comparisons of differences between the means of two treatment groups were performed using the paired two-tailed student's t-test, while comparisons among multiple groups were carried out using one-way or two-way analysis of variance (ANOVA), followed by Tukey's multiple comparisons.The results are expressed as the mean ± SD, and a P value < 0.05 was considered to be statistically significant.

F. nucleatum isolation and characterization
After clinical evaluations of more than 60 cases, 16 patients with periodontitis who met the desired inclusion criteria were identified by the dentist and candidates for sampling.After bacterial culture, 11 samples were excluded from the study due to the mismatch of colony shape, microscopic appearance, and biochemical tests.
Isolates with white-gray speckled colonies that were dry, irregular, crumb-like circular with an entire edge on CA selective plate were presumed to be F. nucleatum (Fig. 1A).Gram-staining for these colonies, confirmed the presence of gram-negative unique spindle shape with tapered ends species (Fig. 1B).The identification of the isolates at the species level was done using biochemical tests.The isolates that were lipase negative (the absence of clear zone around bacterial colonies), indole positive (the formation of a red ring on SIM medium), susceptible to kanamycin, penicillin, and colistin but resistant to vancomycin were considered as F. nucleatum (Fig. 1C-E).
Finally, bacterial ghosts (Heat treated and PFA-fixed) and secretomes were prepared as mentioned above.

Normal and cancerous fibroblast cells out-growth and morphological characterization
Isolated fibroblast cells from normal and cancerous colorectal tissue by explant method, sub-cultured, and after reaching passages3-5, they were used for the next analysis.From the point of morphological view, the elongated, spindle-like shape of cells was observed using light microscopy to represent fibroblast-like cells.NFs are smaller, spindle-shaped, and homogeneous, while CAFs are stellate, with large and multiple nuclei and very granular (Fig. 2).

FAP as a marker that differentiates CAFs from NFs
To compare the expression pattern of FAP and α-SMA in NF and CAF, the flow cytometry analysis was performed.The percentage of cells expressing the FAP marker was significantly higher in the CAF cell population compared to NF cells (*P = 0.016, Fig. 3A, C).Although the expression pattern of the α-SMA marker was heterogeneous in both cells, it was higher in NF cells population than CAF cells; however, this difference was not statistically significant (Fig. 3B, D).

Effect of F. nucleatum components on the expression pattern of FAP and α-SMA by fibroblast cells
To investigate the effect of F. nucleatum components on the fibroblast's phenotype, we examined the expression patterns of FAP and α-SMA in different treatment conditions.

Research
Discover Oncology (2024) 15:292 | https://doi.org/10.1007/s12672-024-01156-0 Considering the heterogeneity of the expression pattern of FAP and α-SMA markers in CAF and NF cells of different patients, the results of the effect of different treatments represented a relative fold change between treatment and control for each patient (Table 2).
The flow cytometry analysis showed that concerning the expression of the two mentioned markers followed by the treatment with different F. nucleatum components, isolated fibroblast obtained from normal and cancerous colon tissue, do not behave differently and in general, treatment with different bacterial components did not lead to significant changes in the expression of FAP and α-SMA markers (Fig. 4).

Comparison of IL-6 and TGF-β levels in the supernatant of fibroblast cells before and after treatment with F. nucleatum components
To investigate the function of normal and cancerous fibroblast cells before and after treatment with F. nucleatum components, the concentration of IL-6 and TGF-β cytokines in CAFs and NFs conditioned media was assayed by ELISA.Our results showed that the amount of IL-6 in the supernatant of CAF cells before and after treatment in all 4 conditions (untreated, treated with heat-killed and PFA fixed ghosts and secretome) was higher than in NF cells.However, this difference was significant only in the group treated with the heated ghost (*p < 0.05, Fig. 5A).
Comparison of the amount of IL-6 in the 4 mentioned conditions, in the CAFs and NFs group separately showed that the treatment of CAF cells with ghosts significantly increased the secretion of IL-6 compared to the treatment with secretome-treated ones (*p < 0.05, ***p < 0.0005, Fig. 5B).
The amount of TGF-β in the supernatant of CAF cells before and after treatment in all 4 conditions was compared to NF cells, but no statistically significant difference was observed (Fig. 5C) between groups.However, significant overexpression of TGF-β was detected in CAF cells after exposure to PFA-fixed ghost in comparison with heated ghost and untreated groups (*p < 0.05, Fig. 5D).
Due to the heterogeneity between the results, in addition to comparing the average expression by the above methods, each patient's NFs were compared with his CAFs under 4 different conditions using the Paired T-Test.IL-6 levels were significantly higher in the treatment of CAF cells with F. nucleatum components except sectertome compared to NF cells (*p < 0.05, Fig. 5E).Also, no statistically significant difference was observed concerning TGF-β levels in this comparison (Fig. 5F).
Next, we used the One-Way ANOVA test to investigate the effect of different treatments in comparison with each other.In this case, all tumoral and normal tissues were considered as one group and were compared under four conditions.The results of this analysis showed that treatment with two types of ghosts increased the production of cytokine IL-6 compared to the untreated group, but none of them showed significant differences.However, this difference was significant between the groups treated with two types of ghosts compared to the group treated with secretions (*p < 0.05, **p < 0.005, Fig. 5G).Treatment with PFA-fixed ghost and secretions increased the levels of this TGF-β compared to the untreated group; however, the difference was statistically significant only between PFA-fixed ghost treated group compared to the untreated group.Also, the level of TGF-β was significantly higher in the PFA-fixed ghost-treated group compared to the heated ghost-treated group (**p < 0.005, ***p < 0.0005, Fig. 5H).

Comparison of F. nucleatum presence in normal and tumor tissues
To reveal the previous exposure of both normal and tumoral tissues with F. nucleatum, qPCR was conducted.The analysis of the relative nusG gene expression in CRC tissues was conducted in a paired way by the adjacent normal tissue of each sample as its specific calibrator.Among the clinical samples with paired tumor and normal samples, the relative amount of the nusG gene was lower in tumor tissue than in adjacent normal tissue in 4 patients (Fig. 6) but in one patient it was higher in tumor tissue than in adjacent normal tissue.

Discussion
CAFs, as the most crucial and abundant cells in the stroma of solid tumors such as CRC or PDAC are in reciprocal communication with TME components and as a result affects the growth and progression of tumors.Moreover, with the deep study of the interaction mechanism between CAFs and immune cells in the TME, a growing amount of evidence indicates CAFs leading immune cells polarization towards activation or exhaustion.In this mutual interaction, both CAFs and tumor cells are capable of altering each other's functions [10].So, to target CAFs for optimizing tumor therapy, identifying the characteristics of CAFs as well as their functions in the TME is of great importance.Also, identifying the factors that lead to the transformation of NFs into CAFs and the aggressiveness of this cell has a key role, too.
From another point of view, CAFs are known to comprise a diverse cell population consisting of several subtypes [45].Support for this notion comes from recent studies on a variety of cancers, such as PDAC, breast or colon carcinoma [46], oral carcinoma [47], and lung cancer [48], in which functionally distinct subclasses of CAFs were identified by the expression of a limited set of markers [49].Among these markers, α-SMA and FAP are among the most important markers of fibroblast cells, which are involved in a wide range of CAFs activities, including tumor growth and proliferation, inhibition of the immune system, prevention of drug delivery, metastasis, and angiogenesis.Also, available data confirmed FAP is the main marker of CAF activity in CRC [50].
FAP is one of the most strongly expressed genes in the stroma of solid tumors and is upregulated in over 90% of epithelial carcinomas.Due to its high expression in the tumor stroma, numerous studies have used FAP as a marker of activated CAFs [51][52][53][54].
This has resulted in the widespread use of FAP as an identifier of potential CAF populations, typically in combination with other markers [55].FAP is also widely considered one of the most viable CAF markers for potential clinical application.Depletion of the FAP-positive fibroblast population in transgenic mice led to cytokine-mediated hypoxic necrosis of both the tumor and the stroma [28].For this reason, FAP-based therapies, such as FAP-inhibitors and FAP-targeting monoclonal antibodies, have been submitted for clinical trials these days.It has also been shown that targeting FAP using CAR-T cells can effectively increase the killing of tumor cells in vitro [56].Jingjing et al. showed that the cause of desmoplastic TME formation in CRC is the interaction between FAP + CAFs and SPP1 + macrophages, which results in resistance to immunotherapy and further reduces the efficacy of PD-L1 treatment [57].Thus, there is interest in FAP + CAFs as a potential target for anti-tumor treatments, and existing research suggests that FAP-targeted drugs can exert curative effects in models of most solid tumors [29].Moreover, several studies have shown the significance of α-SMA-positive CAFs in development and progression of solid tumors [58].
Fig. 6 Relative quantification of the F. nucleatum in CRC and adjacent normal mucous tissues.The mean relative abundance of F. nucleatum (normalized to PGT expression) in CRC tissue versus adjacent normal tissue of both independent runs is reported for each patient sample.The y-axis represents mean fold gene expression change (2 −ΔΔCt ) while the x-axis represents patient samples In line with the above, one of the main goals of this study was to compare the expression pattern of two markers α-SMA and FAP in CAF and NF cells isolated from colon tissue of patients with CRC.Our results showed that the expression pattern of these markers is different in isolated CAF and NF cells.The expression of α-SMA in normal and cancerous fibroblasts was not significantly different, although in NF it had a higher expression.But the expression of the FAP marker was significantly higher in the CAF cells.
Consistent with our findings other studies concerning the expression pattern of two mentioned markers, α-SMA and FAP, by fibroblast subsets provide similar results.For example, in women with a high prevalence of ductal carcinoma breast cancer, CAFs make up 50 to 70% of the cells in the TME.In this tumor, FAP overexpression was typically observed in the connective tissue, the main location of CAF cells, which led to the conclusion that FAP could be considered a major marker for CAFs.However, normal breast tissue has low and sometimes undetectable levels of FAP expression [29,59].In another study conducted by Yashiro et al., the percentage of NF and CAF cells expressing α-SMA isolated from stomach tissue was estimated at 42% and 18%, respectively, confirming the higher expression of α-SMA marker in NF cells than in CAF cells [60].However, according to the expression intensity, FAP Hi and α-SMA Hi fibroblasts, respectively form different populations of CAF and NF cells in tumors derived from epithelial cells [61].
Another important finding of our study was the heterogeneous expression pattern of the two mentioned markers among different patients.So, the percentage of FAP + cells in the CAF population varied from 29 to 67% and in NF cells from 2 to 37%.Also, the percentage of α-SMA + cells in CAF cells of patients varied from 4 to 28% and in NF cells from 4 to 42%.To confirm this finding, we refer to the study on 217 patients with CRC conducted by Mo Son et al. in 2019, that showed the percentage of FAP + CAF and α-SMA + CAFs in patients varied from 0% (in 4 patients) to98% and 23%, respectively [62].Also, during another study in 2020, 92 CRC tissues and 19 normal tissues adjacent to the tumor tissue were examined in terms of FAP marker expression.Eight tumor tissues and four normal tissues were negative in terms of FAP expression, and 84 tumor tissues and 15 normal tissues expressed the FAP marker, of which 72 tumor tissues and 1 normal tissue showed a high level of expression of the FAP marker at the surface of CAFs [61].Therefore, according to the available data, although FAP can be a marker for targeting CAFs and as a result TME, due to its heterogeneous expression, FAP targeting in tumors should be based on the expression pattern of this marker.
After conducting various studies, it was found that a part of this heterogeneity is explained by the fact that CAFs originate from different cells [63,64].Another part of the heterogeneity of CAFs is related to their different function in TME.So, some CAFs secrete some growth factors such as FGF, IL-6, and IL-8, which cause the growth and expansion of cancer cells and the polarization of immune cells towards exhausted cells.Meanwhile, in the context of drug resistance, it is approved that CAFs activate the JAK-STAT3 and PI3K-Akt pathways in cancer cells by secreting IL-6, inducing the upregulation of epithelial − mesenchymal transition (EMT) and E3 ubiquitin ligase complex function [30,65].
The secretome analysis of separated fibroblasts in our study showed the expression of IL-6 by CAF cells was higher than that of NF cells, but TGF-β produced in both groups of CAF and NF cells was low and did not differ significantly from each other.In this regard, Ohlund and his colleagues reported that CAFs with a low expression level of the α-SMA marker produce a large amount of IL-6, and CAFs that express a high amount of FAP and α-SMA produce more TGF-β that both subtypes play an important role in tumor progression [66].Another study on pancreatic cancer showed that α-SMA + CAF cells produce macrophage colony-stimulating factor 1 (M-CSF-1), IL-6, TGF-β, and CCL2 to promote monocyte recruitment and macrophage polarization into M2 macrophages [24].
Microbiome plays a significant role in the complex media of CRC microenvironment and its critical role in the development and progression of the tumor and immune system function has been indicated [67].
One of the most important components of the microbiome is F. nucleatum.Various studies have shown that cell surface and intracellular proteins, and microbial and cell secretions of this bacteria, which include exosomes, growth factors, cytokines, and secreted DAMPs can affect the TME [66,68,69].Also, specifically, the role of secretions and proteins of F. nucleatum has been proven in tumor formation and development [70].In this regard, one of the goals of this study was to investigate the effect of F. nucleatum components on the function and phenotype of fibroblast cells.For this purpose, the effect of bacterial secretions and ghosts on the percentage of α-SMA and FAP and IL-6 and TGF-β production were evaluated.
Following the treatment of CAfs and NFs to the heat-killed ghosts, the expression of the α-SMA marker in CAF cells was often increased compared to the control group, although it was not significant.However, this increasing pattern was not observed in NF cells.Therefore, it can be assumed that the killing of bacteria and the spilling out of proteins and internal bacterial components such as genome and LPS have an effect on increasing the expression of α-SMA marker in CAF cells, which can potentially affect the inhibitory functions of CAFs [40,68,[71][72][73][74].

Research
Discover Oncology (2024) 15:292 | https://doi.org/10.1007/s12672-024-01156-0 The treatment of CAF and NF cells with PFA-fixed ghosts also showed similar results for α-SMA and FAP markers.This over-expression could be due to the exposure to bacterial surface pathogenic proteins such as FadA, RadD, and Fap2 that could be increased fibroblast activity to produce tumorigenic factors such as α-SMA and FAP markers.In the group treated with secretome, the expression of FAP and α-SMA markers often decreased compared to the control group, although it was not statistically significant, which is probably due to the death of fibroblast cells caused by the lethality of the secretome.
A study has shown during the killing of F. nucleatum, endotoxins, especially lipopolysaccharide (LPS) of the bacteria are released, which are recognized by TLRs on the surface of epithelial cells and fibroblasts.Also, the intracellular danger signals then lead to the activation of NLRP3 and the subsequent release of cytokines such as IL-1β [68].F. nucleatum induces the production of inflammatory cytokines by activating NF-κB through TLR4 receptor signaling and by producing FadA protein that binds to E-cadherin of host epithelial and fibroblast cells, which activates the β-catenin signaling pathway.These functions play an important role in creating oncogenic and inflammatory responses and forming a suitable environment for tumor growth [40,[71][72][73]75].Also, a study has shown that a large number of bacterial virulence factors, which are effective in the initiation and progression of inflammatory bowel diseases, were present in the secretions of F. nucleatum, which can lead to tumorigenesis in host cells [76].
In all treatment conditions, the amount of IL-6 in the supernatant of CAF cells was higher than in NF cells, but it was significant only in the group treated with heated ghosts.The TGF-β expression in the supernatant of CAF and NF cells was not significantly different in any treatment.Also, after comparing the overall effect of the treatments with each other, we observed a significant increase in the production of IL-6 in the group treated with ghosts (heated and PFA-fixed) compared to the group treated with secretome.
About TGF-β, this increase was also observed in the group treated with PFA-fixed ghosts compared to the untreated group.Also, the level of TGF-β production was significantly higher in the group treated with PFA-fixed ghosts compared to the heated-ghost treated group.
In this regard, previous studies showed that the presence of F. nucleatum in the intestines of people with CRC stimulates the inflammatory response, which leads to the ROS production, the release of inflammatory factors such as COX-2, IL-1β, IL-6, IL-8, and TNFα from the cells of the TME, and epigenetic changes in cancer cells [42,73].In 2019, Wu et al. stated that F. nucleatum binds to intestinal epithelial cells and fibroblasts by its surface proteins, including FadA, Fap2, and RadD, and induces the migration of inflammatory cells and the production of inflammatory factors.In addition, F. nucleatum can inhibit the intestinal immune system by suppressing the function of immune cells such as macrophages, T cells, and NK cells and contribute to the development of CRC [70].Li and his colleagues also have indicated F. nucleatum isolated from the mouth affects cancer cells in oral squamous cell cancer, causing them to produce IL6 and IL8.These cytokines affect CAFs and lead to the activation of the JAK/STAT pathway in CAF cells and ultimately induce the production of VEGF and tumor angiogenesis [66].

Importance
According to the importance of Fusobacterium nucleatum as well as cancer associated fibroblasts in the induction and progression of colorectal cancer, this is the first study preliminary investigate regarding the effect of F. nucleatum on the function and phenotype of isolated fibroblast cells.

Conclusion
According to our findings, α-SMA and FAP expression on CAF and NF is variable and probably influenced by TME condition.Also, the higher expression of FAP on the surface of CAF cells compared to NF cells besides the important role of FAP in tumor development and immune system suppression during other studies indicates the importance of this marker in tumor development, so that it can be considered as a therapeutic target in the tumor.However, due to its heterogeneous expression, FAP targeting in tumors should be based on the expression pattern of this marker.Furthermore, it should be noted that because FAP expresses on the surface of other cells such as macrophages, identification and targeting of other CAF-specific markers should be considered.In addition, due to the association of FAP with worse prognosis, invasion, metastasis, and treatment resistance in several cancers, the expression level of the FAP marker can be used as a biomarker in determining prognosis, recurrence, and diagnosis of tumor status.

Fig. 1 Fig. 2
Fig. 1 characterization of isolated F. nucleatum.A Dry, irregular, crumb-like circular colonies of F. nucleatum on Columbia agar plate.B Morphological confirmation andgram staining of isolated bactria, F. nucleatum is gram-negative with a long, thin, spindle-shaped appearance in gram staining, (× 100).C and D) biochemical characterization of isolated strain (C) The absence of a clear zone around bacterial colonies indicates the negativity of the lipase test, D the formation of a red ring on the SIM medium indicates the positivity of the indole test for isolated strain, E the presence of inhibition zones around kanamycin, penicillin, and colistin antibiotic indicates the sensitivity of F. nucleatum while the absence of inhibition zones around the vancomycin indicates the resistance of the F. nucleatum to it.KM kanamycin, PCN penicillin, VAN vancomycin, COL colistin

Fig. 3
Fig. 3 Normal and cancerous fibroblast isolated from colon tissue differentially expressed FAP and α-SMA.Representative flow cytometry data showed the frequency of α-SMA + and FAP.+ fibroblastic cells isolated from normal (A) and cancerous colon tissue (B).Comparison of the percentage of cells expressing FAP (C) and α-SMA (D) among normal and cancerous fibroblastic cells, (*p < 0.05)

Fig. 4 Fig. 5
Fig. 4 Effect of the F. nucleatum on the expression of FAP and α-SMA by fibroblast cells.Representative Flow cytometry data of fibroblasts from the tumor (top) and normal (bottom) tissue in coculture with F. nucleatum based on the expression of two markers, FAP and α-SMA.The gating of cells in an FSC vs. SSC plot to separate fibroblast cell populations based on size and granularity (A), unstained tube (B), cells expressing FAP and α-SMA markers in the control (untreated) group (C), the group treated with heat-killed ghosts (D), the group treated with PFA-fixed ghosts (E) and the group treated with secretome of F. nucleatum.(F)

Table 2 The
percentage of NF and CAF cells expressing α-SMA and FAP markers in the groups treated with F. nucleatum components relative to the control group sample α-SMA in control group (%) | https://doi.org/10.1007/s12672-024-01156-0